A mixture of: N,N-di(hydrogenated alkyl C14-C18)phthalamic acid; dihydrogenated alkyl (C14-C18)amine

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 26th July 2010 and 21st October 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 15th September 2009; Date of signature: 26th November 2009

Test material

Test material information

Constituent 1

Reference substance name:

TAB 2V

IUPAC Name:

TAB 2V

Details on test material:

Sponsor's identification: TAB-2V
Description: pale yellow flakes
Batch number: 5028178
Date received: 7 June 2010
Expiry date: 7 June 2012
Storage conditions: room temperature in the dark

The integrity of supplied data relating to the identity, purity and stability of the test item is the responsibility of the Sponsor.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

The test item concentration in the test samples was determined by high performance liquid chromatography mass spectrometry (HPLC-MS) using an external standard. The test item gave a chromatographic profile consisting of a number of un-resolved peaks. The results have been calculated using the total peak area associated with the test item. The method was developed by the Department of Analytical Services, Harlan Laboratories Ltd, Shardlow, UK.

Standard solutions of test item were prepared in tetrahydrofuran at nominal concentrations of 0.10, 0.20, 0.50, 1.0, 2.0 and 5.0 mg/l.

The standards and samples were analysed by HPLC-MS using the following conditions:

HPLC-MS System: Agilent Technologies 1200 MSD incorporating autosampler and workstation.
Mass selective detector -
Source: electrospray
Fragmentation energy: 150 volts
Polarity: positive
Mode: single ion mode with selected ions at 466, 494 and 522 m/z
Gas temperature: 350°C
Drying gas: 11 litre/minute
Nebuliser pressure: 55 psi
Capillary voltage: 4000 volts
Gain: 1
Column: Luna phenyl-hexyl, 3IJm (50 x 2.0 mm id)
Column temperature: 60°C
Mobile phase: 2- propanol: ultra gradient water: formic acid (60:40:0.1 v/v/v)
Flow rate: 0.5 ml/min
Injection volume: 100 µl
Retention time: approximately 1.5 to 2 minutes

Test solutions

Vehicle:

no

Details on test solutions:

An amount test item (250 mg) was added to the surface of 2.5 litres of culture medium to give the 100 mg/l loading rate. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 95 hours and the mixture allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 em from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 ml discarded) to give the 100 mg/l loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present.

An aliquot (2 litres) of the loading rate WAF was inoculated with algal suspension (25.9 ml) to give the required test concentration of 100 mg/iloading rate WAF. The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: CCAP 276/20
- Source: The Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland

Prior to the start of the test sufficient master culture was added to approximately 100 ml volumes of culture media contained in conical flasks to give an initial cell density of approximately 103 cells/ml. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 - 150 rpm) and constant illumination at 24 ± 1°C until the algal cell density was approximately 104 - 105 cells/ml.

ACCLIMATION
- Acclimation period: not applicable
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: There were no abnormalities detected in any of the control or test cultures.

Study design

Test type:

not specified

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Test temperature:

24 ± 1 °C
The temperature within the incubator was recorded daily.

pH:

The pH values of the control cultures were observed to increase from pH 7.3 - 7.4 at 0 hours to pH 7.7 - 7.8 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

The pH of each control and test flask was determined at initiation of the test and after
72 hours exposure. The pH was measured using a WTW pH 320 pH meter.

Nominal and measured concentrations:

Control, 100 mg/l, the measured concentrations were < LOQ.
There were 6 replicates per dose.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250ml conical flasks
- Type (delete if not applicable): closed
- Aeration: the master culture was constantly aerated, the test vessel was closed and shaken continuously, but not aerated.
- Initial cells density: 4.08E+03 (mean of the test group)
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
The culture medium was prepared using reverse osmosis purified deionised water (Elga Optima 15+) and the pH adjusted to 7.5 ± 0.1 with 0.1 N NaOH or HCI.

Culture medium:

NaN03 - 25.5 mg/I
MgCI2.6H20 - 12.164 mg/I
CaCI2.2H20 - 4.41 mg/I
MgS04.7H20 - 14.7 mg/I
K2HP04 - 1.044 mg/I
NaHC03 - 15.0 mg/I
H3B03 - 0.1855 mg/I
MnCI2.4H20 - 0.415 mg/I
ZnCI2 - 0.00327 mg/I
FeCI3.6H20 - 0.159 mg/I
CoCI2.6H20 - 0.00143 mg/I
Na2Mo04.2H20 - 0.00726 mg/I
CuCI2.2H20 - 0.000012 mg/I
Na2EDTA.2H20 - 0.30 mg/I
Na2Se03.5H20 - 0.000010 mg/I


TEST MEDIUM / WATER PARAMETERS
- Total organic carbon:
Pre-study work indicated that there was a significant increase in the amount of total organic carbon by extending the preparation period from 24 to 96 hours. As such for the purposes of the definitive test the test item was to be prepared as a Water Accommodated Fraction (WAF) using a 95-Hour stirring period followed by a 1-Hour settlement period.
- Culture medium different from test medium: no
- Intervals of water quality measurement: no data

OTHER TEST CONDITIONS
- The flasks were kept at 24 ± 1°C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 - 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
- The vortex depth was recorded at the start and end of the mixing period and was observed to have formed a dimple at the media surface.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.
- Other:
A Student's t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) was carried out on the growth rate and yield data after 72 hours for the control and the 100 mg/l loading rate to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

TEST CONCENTRATIONS
The Beckman Coulter® counts indicated that the test item had no effect on algal growth at the test concentrations of 10 and 100 mg/l loading rate WAF. As such it was considered appropriate to conduct the definitive test at a single loading rate of 100 mg/l to confirm that no effect on algal growth was observed.

Reference substance (positive control):

yes

Remarks:

The positive control was conducted between 21 June 2010 and 2 July 2010. A positive control (Harlan Laboratories Ltd Project No: 0039/1161) used potassium dichromate as the reference item at concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/I.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

dissolved

Remarks:

(loading rate WAF)

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

dissolved

Remarks:

(loading rate WAF)

Basis for effect:

growth rate

Results with reference substance (positive control):

- Results with reference substance valid? Yes - The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Exposure of Desmodesmus subspicatus (CCAP 276/20) to the reference item gave the
following results:
ErC50 (0 - 72 h) = 0.74 mg/I*
EyC50 (0 - 72 h) = 0.37 mg/I, 95% confidence limits 0.34 - 0.41 mg/I

No Observed Effect Concentration (NOEC) based on growth rate = 0.25 mg/
No Observed Effect Concentration (NOEC) based on yield = 0.25 mg/I
Lowest Observed Effect Concentration (LOEC) based on growth rate = 0.50 mg/I
Lowest Observed Effect Concentration (LOEC) based on yield = 0.50 mg/I

Reported statistics and error estimates:

Inhibition of growth rate:
Statistical analysis of the growth rate data was carried out for the control and 100 mg/l loading rate WAF test group using a Student's t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981). There were no statistically significant decreases in growth rate (P~0.05), between the control and 100 mg/l loading rate WAF test group and therefore the "No Observed Effect Loading Rate" (NOEL) based on growth rate was 100 mg/iloading rate WAF.

Inhibition of yield:
There were no statistically significant decreases in yield between the control and 100 mg/I loading rate WAF (P20.05) and, therefore the "No Observed Effect Loading Rate" (NOEL) based on yield was 100 mg/lloading rate WAF.

Any other information on results incl. tables

Table 1 - Cell Densities and pH Values in the DefinitiveTest

Nominal Loading Rate (mg/l)		pH	Cell Densities*(cells per ml)				pH
		0 h	0 h	24 h	48 h	72 h	72 h
Control	R1	7.4	4.07E+03	1.44E+04	4.80E+04	1.49E+05	7.8
	R2	7.4	4.11E+03	1.11E+04	4.56E+04	1.32E+05	7.8
	R3	7.4	4.02E+03	1.18E+04	4.27E+04	1.41E+05	7.7
	R4	7.3	4.03E+03	1.14E+04	3.22E+04	1.54E+05	7.7
	R5	7.3	4.10E+03	1.27E+04	2.89E+04	1.33E+05	7.7
	R6	7.3	4.09E+03	1.22E+04	3.61E+04	1.41E+05	7.7
	Mean		4.07E+03	1.23E+04	3.89E+04	1.42E+05
100	R1	7.3	4.06E+03	1.37E+04	4.79E+04	1.93E+05	7.6
	R2	7.3	4.12E+03	1.08E+04	3.66E+04	1.80E+05	7.7
	R3	7.3	4.10E+03	7.11E+03	3.83E+04	1.56E+05	7.7
	R4	7.3	4.10E+03	9.47E+03	3.50E+04	2.04E+05	7.7
	R5	7.3	4.07E+03	1.12E+04	3.71E+04	1.40E+05	7.7
	R6	7.3	4.05E+03	1.30E+04	4.35E+04	1.87E+05	7.7
	Mean		4.08E+03	1.09E+04	3.97E+04	1.77E+05

 

Table 2 - Daily Specific Growth Rates for the Control Cultures in the Definitive Test

Control	Daily Specific Growth Rate (cells/ml/hour)
	Day 0 - 1	Day 1 - 2	Day 2 - 3
R1	0.054	0.050	0.047
R2	0.042	0.059	0.044
R3	0.045	0.054	0.050
R4	0.044	0.043	0.065
R5	0.048	0.034	0.063
R6	0.047	0.045	0.057
Mean	0.047	0.048	0.054

Table 3 - Inhibition of Growth Rate and Yieldin the Definitive Test

Nominal Loading Rate (mg/l)		Growth Rate (cells/ml/hour)		Yield (cells/ml)
		0 – 72 h	% Inhibition	0 – 72 h	% Inhibition*
Control	R1	0.050		1.45E+05
	R2	0.049		1.28E+05
	R3	0.049		1.37E+05
	R4	0.051	-	1.50E+05	-
	R5	0.049		1.29E+05
	R6	0.049		1.37E+05
	Mean	0.050		1.38E+05
	SD	0.001		8.67E+03
100	R1	0.054	[8]	1.89E+05
	R2	0.053	[6]	1.76E+05
	R3	0.051	[2]	1.52E+05
	R4	0.055	[10]	2.00E+05
	R5	0.049	2	1.36E+05
	R6	0.053	[6]	1.83E+05
	Mean	0.053	[5]	1.73E+05	[25]
	SD	0.002		2.40E+04

*Cell densities represent the mean number of cells per ml calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

R₁- R₆= Replicates 1 to 6

R₁- R₆= Replicates 1 to 6

*In accordance with the OECD test guideline only the mean value for yield is calculated

R₁– R₆= Replicates 1 to 6

SD= Standard Deviation

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Desmodesmus subspicatus has been investigated and gave EL50 values of greater than 100 mg/l loading rate WAF (Water Accommodated Fraction). Correspondingly the No Observed Effect Loading Rate was 100 mg/l loading rate WAF.

Executive summary:

Introduction:

A study was performed to assess the effect of the test item on the growth of the green alga Desmodesmus subspicatus. The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) 440/2008.

Methods:

Desmodesmus subspicatus was exposed to a Water Accommodated Fraction (WAF) of the test item, at a single nominal loading rate of 100 mg/l (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1cC. Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

Results:

Exposure of Desmodesmus subspicatus to the test item gave EL*50 values of greater than 100 mg/l loading rate WAF and correspondingly the No Observed Effect Loading Rate was 100 mg/l loading rate WAF. It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/l. Chemical analysis of the test preparations at 0 and 72 hours showed measured test concentrations of less than the limit of quantitation (LOQ) of the analytical method employed were obtained which was determined to be 0.0074 mg/l. This does not infer that no test item was in solution, just that that which was present was at a concentration below the quantifiable limit. Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, and the dissolved test item was below the quantifiable limit of the analytical method, the results were based on nominal loading rates only.